There is an urgent need to control the global epidemic of HIV infection and the development of a vaccine against HIV is one of the major objectives in AIDS research. In general vaccines should activate antigen presenting cells, overcome genetic restriction in T-cell responses and generate T- and B-memory cells. The variability of the viral population poses a further difficulty in obtaining an effective HIV vaccine. A breakthrough in the ongoing attempts to develop a vaccine against AIDS has so far not been reported. It is now generally accepted that an induction of antigen-specific humoral and cell-mediated immunity is crucial for a development of an effective prophylactic and therapeutic vaccine. All three arms of the immune system including neutralizing antibodies, CD8+CTL and T-helper-1 (TH1) cells might be required for protective immunity to HIV. It is known that CTL can clear other viral infections (Ada, Immunol. Cell Biol., 72:447-454, 1994) and that CTL can lyse infected targets early in infection before viral progeny can be produced and released by cell lysis, Ada et al., supra. The focus has been on selection of antigens as well as on design and evaluation of different adjuvances. The antigens used in different in vitro and in vivo studies have all been from crude proteins to various synthetic peptides, mainly from gp160 and to some extent from p24. A large number of studies have been done on the V3 loop of gp120. Induction of both B- and T-cell responses have been observed; however, it has been reported from an in vitro study that a peptide from the conserved region of gp41 has indicated infection enhancement (Bell S. J., et al., Clin. Exp. Immunol., 87 (1): 37-45, (January 1992).
Naturally occurring HIV sequences in vaccine candidates are not capable of stimulating a stable immune response due to the virus's inherent ability to hide by changing the appearance of the epitopes presented on the cell surface of infected cells. The immune system is fooled into believing that a particular amino acid sequence is relevant when in fact the amino acid of importance is hidden.
A recent study of titers of antibodies against the gag p24 protein, has shown that slow progression towards development of AIDS is associated with high titers, while fast progression towards development of AIDS is associated with low titers. It is shown that persons with low p24 antibody titer develop AIDS significantly faster than persons with high p24 antibody tiers (Zwart G., et al. Virology, 201, p. 285-93, June 1994), indicating that p24 can play a key role to control the development of AIDS.
New HIV p24 peptides are described in WO91/13360, wherein the peptides are used in a method of discriminating between a false and true diagnosed HIV-positive serum sample.
Johnson R. P., et al., The Journal of Immunology, Vol. 147, p. 1512-1521, No. 5, Sep. 1, 1991 describe an analysis of the fine specialty of gag-specific CTL-responses in three HIV-1 seropositive individuals. The gag-specific CTL-responses were found to be mediated by CD3+CD8+ lymphocytes which are HLA class I restricted.
EP-A-0 356 007 discloses antigenic determinants, in particular it relates to synthetic polypeptide sequences which are related to proteins present in the HIV-1 and which can be used as a basis for a potential vaccine against AIDS.
Rosenberg E. S. et al., Science, Vol. 278, 21 Nov. 1997, p. 1447-1450 describe that virus specific CD4+ T helper lymphocytes are critical to the maintenance of effective immunity in a number of chronic viral infections, but are characteristically undetectable in chronic human immunodeficiency virus-type 1 (HIV-1) infection. HIV-1-specific proliferative responses to p24 were inversely related to viral load. They conclude that the HIV-1-specific helper cells are likely to be important in immunotherapeutic interventions and vaccine development.
EP 0 230 222, EP 0 270 114, DE 37 11 016 and GB 2 188 639 all in the name of F. Hoffmann-La Roche & Co. Aktiengesellschaft concern recombinant expression and purification of an HTLVIII Gag/Env gene protein or fusion proteins. The proteins consisting of native sequences can be purified to homogeneity and used as a basis for diagnostic tests for detection of antibodies against viruses associated with AIDS. The gag/env protein may also be formulated for use as a vaccine for protection against AIDS through prophylactic immunization.
From a diagnostic and therapeutic point of view, the major problem with using p24 as part of an assay or therapy is associated with the high number of epitopes on p24 which stimulates production of a large number of antibodies with poor specificity, which through repeated boostering on potential mutated sequences can create autoantibodies (Autoantibodies to the alfa/beta T-cell receptors in HIV infection; dysregulation and mimicry. Lake D. F., et al., Proc. Nalt. Acad. Sci. USA, (23): 10849-53, Nov. 8 1994). Further, it is reported that the p24 antibody titer does not reach the same high levels as for the envelope proteins (gp120 and gp41). Normally antibodies to p24 are developed in the early phase of the infection, but the titer is fairly quickly stabilized after the initial infection period. Later the p24 titer gradually decreases while the opposite happens with gp160. These findings can also be seen in relation to recent reports stating that cytotoxic T-cell activity is antagonized by naturally occurring HIV-1 gag variants (Klenerman, P., et al., Nature, 2:369 (6479), p. 355, 2 Jun. 1994). This can be one of the reasons why a rapid stabilization of the p24 titer is seen and why it later starts to decrease.
Based on the above background data, we decided to investigate the possibility of designing novel synthetic peptides which can mimic the p24 epitope without antagonizing the cytotoxic T-cell activity, in order to meet the need for an effective prophylactic and therapeutic vaccine.
The initial work was based on one epitope which was published by Korber B., et al., Human Retroviruses and AIDS 1997 Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, N. Mex. The amino acid sequence of this epitope (203-222) was:
KALGPGATLEEMMTACQGVG(SEQ ID NO:26) RRMRTK SIKD LSSS R R(SEQ ID NO:27)  G  VR       V(SEQ ID NO:28)  S  AA(SEQ ID NO:29)     SE(SEQ ID NO:30)     QQ(SEQ ID NO:31)
The one letter as well as the three letter codes defining the amino acids in the sequences given throughout this specification are in accordance with International standards and given in textbooks, for instance Lehninger A. L., <<Principles of Biochemistry>>, Worth Publishers Inc., New York, 1982. The amino acids given below the head sequence represent the natural variation of the sequence. An initial study of a sequence containing this modified epitope was conducted on the sequence:
wherein X indicates 2-aminohexanoic acid, and the cysteine residues are in an oxidized state, i.e. are forming an intrachain disuiphide bridge. The results (unpublished) from studies using this peptide as part of a diagnostic kit showed that the specificity became 87% (n=279) on a preselected panel of African sera. The sensitivity was surprisingly 100% on a panel of HIV-1 positive sera including HIV-1 subtype O sera, which is quite different from the other subtypes.
In order to improve specificity, i.e. define the amino acids which contribute to a pure non-crossreacting antibody response, a similar study was applied to a significantly shorter and further modified peptide:
wherein X has the above-mentioned meaning and the cysteine residues are forming an intrachain disulphide bridge.
The results from this study showed that the specificity of the assay increased to 96%, and (n=293) which is similar to the specificity obtained in the assay without using the p24 peptide. With a specificity of 87% to the assay where the first peptide was included, it would be likely that the peptide would induce an immune response to more than one epitope since it was recognized by unspecific antibodies, if it was used as a vaccine candidate. The latter, however, shows that the peptide sequence is picking up an immune response which is unique to HIV-1. Consequently, if a sequence based on this is used as an antigen in a vaccine candidate, it would most likely boost a unique immune response to HIV-1.
To further increase the number of T-cell epitopes and reduce the probability for development of escape mutants, three additional peptide sequences were based on the following three sequences from residues 264-284, 253-271 and 166-186, respectively, published in Human Retroviruses and AIDS 1997; A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos:
RWIILGLNKIVRMYSPTSILD(SEQ ID NO:34) KGVVM    M K  C VG  E(SEQ ID NO:35) D M V    V    Q I   G(SEQ ID NO:36)                 S(SEQ ID NO:37)                 A(SEQ ID NO:38) NNPPIPVGEIYKRWIILGL(SEQ ID NO:39) S QAV  KDMLRKGMVM(SEQ ID NO:40) G GSN   KV  D V V(SEQ ID NO:41) H  GT(SEQ ID NO:42) A(SEQ ID NO:43) P(SEQ ID NO:44) and PEVIPMFSALSEGATPQDLNT(SEQ ID NO:45)  RITTTLTE AD  ISYNIYM(SEQ ID NO:46)   LN   AL     V H V I(SEQ ID NO:47)        M      L     A(SEQ ID NO:48)                     V(SEQ ID NO:49)Several modified peptides have been synthesized in order to determine unique sequences which are both specific and sensitive towards HIV-1.